The protein expression group functions primarily as a support group for the principle investigators at the NIEHS. Our focus is to provide the other investigative groups at the NIEHS with a means to generate the protein of interest in a system so that they can perform their experiments. During the course of the last year we have worked on more than 40 such project ranging from heterologous expression in E.coli to produce enough correctly folded protein for x-ray crystal trials to scaling up the growth of a stable mammalian cell line for membrane preparations to test the function of ion transporters. [unreadable] Each new protein that is expressed in E. coli is tested using five different n-terminal tags (6 x His, 6 x His-Thioredoxin, 6 x His-glutathione S-transferase, 6 x His-Maltose binding protein, 6 x His-NusA). Initial expression test are done in Rosetta2(DE3)pLacI cells at 18 C and 30 C. These tags help to fold/solubilize the protein of interest and provide an uniform initial purification step that helps to identify which combination of tag and temperature yield the most of the desired product. The Rosetta cell line helps remove any codon bias against expression in E. coli. This is not the only cell line that does this, and we are not endorsing this cell line over the others. Once expression an n-terminal tag is selected, other variables such as alternative cells lines, media and temperature can all be optimized to give the best yield.[unreadable] If expression in E. coli fails to work or is not feasible due to the need for post translational modifications, then baculovirus/ insect cell expression is tried first. Expression is investigated using three n-terminal tags (6 x His, 6 x His-glutathione S- transferase, and 6 x His-Maltose binding protein) and two cell lines (SF9 and High Five). Expression trials are carried out at both 27 C and 20 C. In all cases, co-expression of green fluorescent protein driven by a different promoter is used to help titer the baculovirus and demonstrate that infection was successful.[unreadable] The protein expression group also has vectors available for expression of protein in mammalian cell lines. Expression is tested in Cos-7, HEK293, CHO and Hela cells unless a more unique cell line is desired by the principle investigator. Expression is first tested transiently followed by the generation of a stable cell line, if this is possible. [unreadable] If the above methods fail to yield a protein that in other that insoluble aggregates (inclusion bodies), then protein refolding is attempted. Each refolding project is tested using both rapid dilution and a high hydrostatic pressure approach. The rapid dilution approach is performed in a 96 well multi matrix format while the high hydrostatic approach uses a more sequential multi-sample (20 sample per run) approach.[unreadable] [unreadable] Project examples:[unreadable] [unreadable] P-Selectin project- Reyes-Reyes, E.M. and Akiyama, S.K.: Cell-surface Nucleolin is a Signal Transducing P-selectin Binding Protein for Human Colon Carcinoma Cells,Exp. Cell Res, 314: 2212-223, 2008.[unreadable] The Akiyama group had previously shown that P-selectin binding to Colo-320 human colon carcinoma cells induces specific activation of the a5b1 integrin with a concomitant increase of cell adhesion and spreading on fibronectin substrates in a phosphatidylinositol 3-kinase (PI 3-K) and p38 MAPK-dependent manner. Building on these studies, they identified by affinity chromatography and characterized nucleolin as a P-selectin receptor on Colo-320 cells. Nucleolin mAb D3 decreases significantly the Colo-320 cell adhesion to immobilized P-selectin-IgG-Fc. Moreover, nucleolin becomes clustered at the external side of the plasma membrane of living, intact cells when bound to cross-linked P-selectin-IgG-Fc chimeric protein. In addition, they demonstrated that P-selectin binding to Colo-320 cells induced tyrosine phosphorylation specifically of cell-surface nucleolin and formation of a signaling complex containing cell surface nucleolin, PI 3-K, and p38 MAPK. Using siRNA approaches, it was shown that both P-selectin binding to Colo-320 cells and formation of the P-selectin-mediated p38 MAPK/ PI 3-K signaling complex require nucleolin expression. These results show that nucleolin (or a nucleolin-like protein) is a signaling receptor for P-selectin on Colo-320 cells and suggest a mechanism for linkage of nucleolin to P-selectin-induced signal transduction pathways that regulate the adhesion and the spreading of Colo-320 on fibronectin substrates.[unreadable] [unreadable] The protein expression group developed the protocols for increased production of p-selectin to allow the project to move forward.[unreadable] [unreadable] Rhox13 project: Edward M Eddy[unreadable] Homeobox genes encode transcription factors whose expression organizes programs of development. A number of homeobox genes expressed in reproductive tissues have been identified recently, including a colinear cluster on the X chromosome in mice. This has led to an increased interest in understanding the role(s) of homeobox genes in regulating development of reproductive tissues including the testis, ovary, and placenta. The Eddy group identified and characterized a novel homeobox gene of the paired-like class on the X chromosome distal to the reproductive homeobox (Rhox) cluster in mice. Transcripts are found in the testis and ovary as early as 13.5 days post coitum (dpc). Transcription ceases in the ovary by 3 days post partum (dpp), but continues in the testis through adulthood. The Rhox13 gene encodes a 25.3 kDa protein expressed in the adult testis in germ cells at the basal aspect of the seminiferous epithelium.[unreadable] [unreadable] The protein expression group expressed the Rhox13 gene in E.coli and purified it. This protein was used to confirm the identity of the protein in the native cells used in these studies.[unreadable] [unreadable] hPol Nu project: Thomas Kunkel [unreadable] Human DNA polymerase Nu (Pol Nu) is a conserved family A DNA polymerase of unknown biological function. Physical and biochemical characterization aimed at understanding Pol Nu function is somewhat hindered by the fact that when over expressed in E. coli, Pol Nu is largely insoluble, and the small amount of protein that is soluble is difficult to purify. To obtain soluble Pol Nu for future studies, high hydrostatic pressure was used to solubilize and refold active Pol Nu from inclusion bodies. Active Pol Nu can be refolded and purified. The properties of the refolded enzyme are comparable to those of the small amount of Pol Nu that can be purified from the soluble fraction. The approach used here may be applicable to other DNA polymerases that are insoluble when expressed in E. coli.[unreadable] [unreadable] hAPP685 project: Elucidation of O-glycosylation structures of the -amyloid precursor protein by liquid chromatography mass spectrometry using electron transfer dissociation and collision induced dissociation. Kenneth Tomer[unreadable] Accumulation and deposition of -amyloid peptide to form neuritic plaques are hallmarks of Alzheimers disease (AD) and AD-related neurodegenerative diseases. -amyloid (A) is derived from the proteolytic cleavage of amyloid precursor protein (APP), a transmembrane protein present in three major isoforms in brain comprising 695, 751 and 770 amino acids, respectively. Among other post-translational modifications, APP is modified during maturation by N- and O- glycosylation, which are thought to be responsible for its expression and secretion. Unlike N-glycosylation, no sites of O-glycosylation of APP have previously been reported. The Tomer group identified three specific O-glycosylation sites of the secreted APP695 (sAPP695) produced in CHO cells. [unreadable] [unreadable] The protein expression group optimized the expression of and purification of the hAPP695 from CHO cells that enabled the study to be performed.